PROJECT SUMMARY. Several new viral respiratory tract infectious diseases with epidemic potential that threaten global health security have emerged in the past 20 years. Influenza A viruses (IAVs) comprise 50% of the emerging respiratory viruses and can cause substantial morbidity and mortality. IAVs can infect a diversity of avian and mammalian species, including humans, and have the remarkable capacity to evolve and adapt to new hosts. Despite the tremendous progress made in virology and epidemiology, which subtype or strain of IAV will cause the next outbreak remains unpredictable. Importantly, there is no clinically simulating, pathophysiologically relevant, and readily available in vitro multi-organ system for predicting the pathogenicity of emerging and re-emerging influenza viruses in humans. Recent compelling evidence have revealed opposing roles for two major classes of bone marrow (BM)-produced innate immune cells in shaping the outcome of IAV infection, with neutrophils offering protection and increase in circulating monocytes being associated with increased pathology. Thus, selective mobilization of either of these two distinct cell types in response to pulmonary infection with IAV can indirectly reveal potential pathogenicity of a given viral strain. The overarching goal of this project is to develop a highly innovative, reductionist, yet advanced and complex, physiologically relevant in vitro model of influenza infection in humans utilizing Organ-on-Chip technology in order to predict virulence and infectivity of different IAV strains, by reproducing clinically and in vivo-observed immunological correlates of infection severity. More specifically, we will engineer a first-in-kind fluidically integrated multi- organ system that recreates BM-lung axis, using primary human-derived cells, for real-time analysis of inflammation and leukocyte mobilization in response to influenza challenge. Our central hypothesis is that this dynamic living microsystem can recapitulate differential immune cell mobilization and tissue pathology in response to high-pathogenicity vs. low-pathogenicity IAV infections in vitro. To address the hypothesis, we propose the following specific aims: (1) to engineer a living and hematopoietically active human BM-on-a-Chip and microfluidically link it to a human Lung Small Airway-on-a-Chip that our team has previously developed and characterize homeostatic physiology and organ-organ crosstalk; and (3) to challenge the BM-Lung microsystem with airborne IAVs under rhythmic breathing and reproduce differential leukocyte mobilization and tissue damage in response to distinctly pathogenic viral strains. Such a novel platform holds great potential in emulating and predicting pathogenicity of IAVs (e.g., during outbreaks, pandemics or when presence of a highly virulent strain is speculated), utilizing human cells isolated from desired donor/patient populations, and without needing to adapt the virus for host (as required for some animal studies). In addition, it can considerably accelerate drug development studies by enabling personalized drug efficacy testing and identification of new therapeutic targets.